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Keywords:

  • FVIII:C assays;
  • severe hemophilia A;
  • SSC classification

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Samples and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Summary.  To assess the practicality of the recent Scientific and Standardization committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) recommendations in respect of the classification of hemophilia we distributed samples from three untreated subjects with hemophilia A to 91 UK hemophilia centers (HCs), comprising 20 comprehensive care centers (CCCs) and 71 HCs. Laboratories were requested to perform their routine factor (F)VIII:C assays and to classify the severity of hemophilia. Median values of < 1 U dL−1 were obtained on two samples. However, for each of the two, approximately 30% of laboratories obtained results in the range 1–29 U dL−1 and 1–33 U dL−1 respectively. For one of these samples 17 laboratories diagnosed severe hemophilia despite obtaining FVIII:C levels in the range 1–5 U dL−1. The median FVIII:C for the third sample was 5.8 U dL−1 with a range of 1.5–36 U dL−1. For this sample eight centers diagnosed severe hemophilia. Fifty-four laboratories obtained a result > 5 U dL−1; 21 of these diagnosed mild hemophilia, 31 moderate hemophilia and two severe hemophilia. Results from CCCs were more accurate and more precise than those from HCs. Our results indicate a need for improved standardization of FVIII assays. In the UK there remains a lack of consensus in respect of the laboratory diagnostic criteria for the classification of hemophilia A.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Samples and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The Scientific Subcommittee on Factor VIII and Factor IX of the Scientific and Standardization committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) has recently recommended that the classification of severe, moderate and mild hemophilia A be defined on the basis of biological factor (F)VIII:C levels of < 1, 1–5 and > 5 IU dL−1, respectively, rather than on the clinical severity of affected individuals [1]. In view of the wide interlaboratory variability of FVIII:C assay results among UK hemophilia centers participating in the proficiency testing programme provided by UK National External Quality Assessment Service (UKNEQAS) [2], we have undertaken an exercise designed to determine whether the precision and the accuracy of FVIII:C assays at low levels of FVIII:C would support the viability of the SSC recommendation.

Samples and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Samples and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

After obtaining written, informed consent, blood samples were obtained from three subjects with hemophilia A. Two of these had clinically severe hemophilia with expected FVIII:C levels < 1 IU dL−1, the third had a clinically mild disorder with an anticipated FVIII:C level between 5 and 10 IU dL−1. None had received factor VIII:C replacement therapy for at least 10 days before their donation. Blood was collected into 1/10 volume 0.105 m trisodium citrate, centrifuged at 2000 g for 10 min, buffered with 0.8% HEPES and lyophilized in aliquots. The samples from the two severely affected subjects were coded 01 and 02; the sample from the mildly affected subject was coded 03.

After obtaining their consent to participate in the study the three samples were distributed to 91 hemophilia centers for FVIII:C determination. The participating centers were informed that the samples were obtained from subjects with hemophilia A. They were asked to report their results and to indicate, by means of a provided tick-box, whether their result signified mild, moderate or severe hemophilia A. The participating centers comprised 20 comprehensive care centers (CCCs) and 71 hemophilia centers (HCs). In the UK these designations are recognized by the Department of Health. CCCs provide the highest level of hemophilia care and they function as tertiary referral centers. HCs provide the first level of specialist care for hemophilia. All hemophilia services, whether provided by a CCC or HC, are included in these definitions.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Samples and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Results, interpretations and information on methodology were returned by 82 centers, 20 CCCs and 62 HCs. Eighty centers performed a one-stage FVIII:C assay, one center a two-stage clotting assay and one center a chromogenic assay. Not all centers returned results in international units (IU dL−1) and participant results are therefore given in units (U dL−1). A summary of results and their interpretations is presented in Table 1 and Fig 1a,b,c. Table 2 gives a breakdown of results according to whether centers were HCs or CCCs.

Table 1.  Summary of results
Sample010203
  1. * Including 17 (22%) laboratories reporting FVIII:C levels of 1–5 U dL−1. †Including 14 (18%) laboratories reporting FVIII:C levels of 1–5 U dL−1.

n797778
Median FVIII:C (U dL−1)< 1.0< 1.05.8
Range (U dL−1)0.0–29.00.0–33.01.5–36.0
Interpretation (n)
Severe70 (89%)*68 (88%)8 (10%)
Moderate9 (11%)8 (11%)49 (63%)
Mild0 (0%)1 (1%)21 (27%)
image

Figure 1. The number of centers that obtained a particular factor (F)VIII:C result on the same sample. The classification of hemophilia (mild, moderate or severe) as determined by each center on the basis of their own local assay result is indicated by the shading. Results for samples 01, 02 and 03 are shown in (a), (b) and (c), respectively.

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Table 2.  Percentage of interpretations related to result, for Comprehensive Care Centers (CCC) and Hemophilia Centers (HC)
Reported FVIII:C (U dL−1)Interpretation sample 01 Median FVIII:C < 1 U dL−1Interpretation sample 02 Median FVIII:C < 1 U dL−1Interpretation sample 03 Median FVIII:C 5.8 U dL−1
SevereModerateMildSevereModerateMildSevereModerateMild
< 117 (85%)15 (75%)
1–52 (10%)1 (5%)3 (15%)3 (15%)8 (40%)
> 51 (5%)1 (5%)6 (30%)3 (15%)
< 136 (61%)39 (68%)
1–515 (25%)7 (12%)11 (19%)6 (11%)3 (5%)10 (17%)
> 51 (2%)1 (2%)2 (4%)25 (43%)18 (31%)

Sample 01

Results and interpretations were obtained from 79 centers. The median FVIII:C was < 1 U dL−1 (range 0.0–29 U dL−1). Fifty-three centers reported a FVIII:C level of < 1 U dL−1; five centers reported 1 U dL−1 and 21 centers reported a value > 1 U dL−1. Seventy centers diagnosed severe hemophilia, including 17 centers that obtained FVIII:C levels of 1–5 U dL−1. Nine centers diagnosed moderate hemophilia. The center employing a two-stage assay reported 1 IU dL−1 and the center employing a chromogenic assay reported 2.3 IU dL−1.

Comparison of results and interpretations from CCCs and HCs (sample 01)

Results and interpretations were received from 20 CCCs and 59 HCs. Of the CCCs, 85% reported a FVIII:C level < 1 U dL−1 compared with 61% of the HCs. Of the three CCCs reporting a FVIII:C level of 1–5 U dL−1, two classified this as severe hemophilia. This compares with 22 of 59 HCs that obtained a FVIII:C level in the range 1–5 U dL−1, with 15 of these classifying this as severe hemophilia.

Sample 02

Results and interpretations were obtained from 77 centers. The median FVIII:C was < 1 U dL−1 (range < 1–33.0 U dL−1). Fifty-four centers reported a result < 1 U dL−1; four reported 1 U dL−1 and 19 centers reported a value > 1 U dL−1. Sixty-eight centers diagnosed severe hemophilia A, including 14 centers that obtained FVIII:C levels between 1 and 5 U dL−1. Eight centers diagnosed moderate hemophilia A and one diagnosed mild hemophilia A. The center employing a two-stage assay reported 1 IU dL−1 and the center employing a chromogenic assay reported 2.2 IU dL−1.

Comparison and interpretations of results from CCCs and HCs (sample 02)

Seventy-five percent of CCCs reported a FVIII:C level < 1 U dL−1 compared with 68% of HCs. Three of the 20 CCCs reported a FVIII:C level in the range 1–5 U dL−1 and in each instance this was considered to represent severe hemophilia. Two CCCs reported FVIII:C levels > 5 U dL−1. These results compare with 17/59 (30%) of HCs that reported FVIII:C levels 1–5 U dL−1 with one laboratory reporting > 5 U dL−1. Severe hemophilia was the considered diagnosis by 11 of the 17 HCs who reported a FVIII:C level in the range 1–5 U dL−1.

Sample 03

Results and interpretations were obtained from 78 centers. The median FVIII:C was 5.8 U dL−1 (range 1.5–36.0 U dL−1). Eight centers that obtained FVIII:C levels between 1 and 7.6 U dL−1 diagnosed severe hemophilia A, 49 diagnosed moderate hemophilia A and 21 mild hemophilia A. Of those laboratories that obtained FVIII:C levels > 5 U dL−1, two diagnosed severe hemophilia, 31 diagnosed moderate hemophilia and 21 mild hemophilia. The center employing a two-stage assay reported 9 IU dL−1 and the center employing a chromogenic assay reported 6.1 IU dL−1.

Comparison and interpretations of results from CCCs and HCs (sample 03)

Eleven of 20 (55%) of CCCs and 13/59 (22%) of HCs reported FVIII:C levels in the range 1–5 U dL−1, the remainder of the results being > 5 U dL−1. Although the median results were similar for the two groups the precision between CCCs was better than that between HCs. The CCC median was 5.2 U dL−1[coefficient of variation (CV) 36.3%] compared with HC median of 6.0 U dL−1 (CV 64.6%). The interpretation of results was similar for the two groups. Three of 11 CCCs reporting a FVIII:C level of 1–5 U dL−1 considered the diagnosis to be severe hemophilia. The same conclusion was drawn by 3/13 HCs reporting a FVIII:C level 1–5 U dL−1.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Samples and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Although there is general acceptance of the concept of severe, moderate and mild hemophilia, there has been no formal agreement as to whether they should be defined in terms of clinical expression or by results of laboratory assays. The issue is further confused by a lack of consensus with respect to classification by basal FVIII:C/IX:C levels. Following a survey of UK hemophilia centers in 1990, a factor FVIII:C/FIX:C < 2% of normal was considered to be diagnostic of severe hemophilia [3]. In contrast, in the USA, Brettler and Levine [4] considered FVIII:C levels of 1% of normal or less to be diagnostic of severe hemophilia, with moderate hemophilia 1–5% of normal and mild hemophilia > 5% of normal. Another report from the USA defines severe hemophilia by a FVIII/IX level < 0.01 U mL−1, moderate hemophilia by a FVIII/IX level 0.02–0.05 U mL−1 and mild hemophilia by levels > 0.05 U mL−1[5]. More recently, in the UK, a National Service specification for Hemophilia and related conditions drawn up by the Haemophilia Alliance [6] defined severe hemophilia by a FVIII/IX level < 2 IU dL−1, moderate hemophilia by a FVIII/IX level of 2–10 IU dL−1 and mild hemophilia by levels of FVIII/IX > 10 IU dL−1.

The combination of this dual (clinical and laboratory) classification system together with the various laboratory definitions of hemophilia has undoubtedly hindered epidemiological studies of hemophilia and has been a source of confusion in clinical studies. These considerations prompted the Scientific Subcommittee on Factor VIII and Factor IX of the SSC of the ISTH to address the classification of hemophilia and they have recommended that plasma procoagulant levels, rather than clinical bleeding symptoms, be used preferentially for the classification of hemophilia. They further recommended that severe hemophilia be defined by a FVIII/IX level < 0.01 IU mL−1 (< 1% of normal), moderate hemophilia by factor levels 0.01–0.05 IU mL−1 (1–5% of normal) and mild hemophilia by factor levels > 0.05 to < 0.40 IU mL−1 (> 5% to < 40% of normal) [1].

The results of this exercise, undertaken 10 months after the publication of the SSC's recommendations, have highlighted a number of difficulties with the recommended classification. It is reasonable to assume, on the basis of the recommended classification, that two of the donors in this study have severe hemophilia A since median values of < 1 U dL−1 were obtained by the majority of laboratories. However, for these two samples 30% of participants obtained values in the range 1–29 U dL−1 and 1–33 U dL−1, respectively. Excluding the single outlier for each sample, these ranges are 1–4 U dL−1 and 1–7 U dL−1, respectively. It is also of some concern that for one of the samples, 17 laboratories diagnosed severe hemophilia despite obtaining FVIII:C levels in the range 1–5 U dL−1. For the other sample, 14 laboratories diagnosed severe hemophilia with FVIII:C results in the range 1–5 U dL−1.

The median FVIII:C for the third sample was 5.8 U dL−1 with a range of 1.5–36 U dL−1. It was surprising, therefore that eight centers (including three CCCs) diagnosed severe hemophilia.

It is clear that some uncertainty exists with respect to hemophilia classification for factor assay results > U dL−1. Fifty-four laboratories obtained such a result but only 21 of these diagnosed mild hemophilia. Of the others, two diagnosed severe hemophilia and 31 moderate hemophilia.

We also performed separate analyses of the results obtained from CCCs and HCs. With respect to the samples obtained from the two subjects with severe hemophilia, our results demonstrate that significantly more CCCs returned FVIII:C levels < 1 U dL−1 than did the HCs. Although laboratories from both groups considered the diagnosis to be severe hemophilia with FVIII:C levels in the range 1–5 U dL−1, this anomaly was much more prevalent with the HCs. With respect to the results obtained on the sample from the subject with moderate or mild hemophilia, although median values were similar for the two groups the variability of results was greater for the HCs.

Although only a single center deployed a chromogenic assay, it is of note that it obtained results of 2.3 and 2.2 U dL−1 on the samples from the two subjects with severe hemophilia.

These results highlight two problem areas with respect to the adoption of the recommended classification of hemophilia. The failure of approximately 30% of laboratories to obtain a FVIII:C result < 1 U dL−1 for two different samples, together with the spread of results from all three samples, strongly suggests the necessity for some improvement in the standardization of FVIII assays. It is reassuring that the results returned by the CCCs were more accurate and more precise than those returned by the smaller HCs. If these differences persist then consideration may be necessary of the provision of diagnostic reference laboratories.

Our results also indicate that at least in the UK, and even among CCCs, the SSC recommendations in respect of the classification criteria have not been unanimously adopted. We are unaware of the underlying reasons for this. There can be no doubt that a unified approach to the classification of hemophilia is highly desirable. This will be achieved only through improved standardization of factor assays and the global acceptance of international definitions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Samples and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We are indebted to the hemophilia center Directors who participated in this study.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Samples and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • 1
    White, GC, Rosendaal, F, Aledort, LM, Lusher, JM, Rothschild, C, Ingerslev, J. Definitions in Hemophilia. Recommendations of the Scientific Subcommittee on FVIII and Factor IX of the SSC of ISTH. Thromb Haemost 2001; 0: 560.
  • 2
    Preston, FE, Kitchen, S. Quality control and FVIII assays. Haemophilia 1998; 4: 6513.
  • 3
    Rizza, CR. Haemophilia and related inherited coagulation defects. In: Bloom, AL, Forbes, CD, Thomas, DP, Tuddenham, EGD, eds. Haemostasis and Thrombosis, 3rd edn. Edinburgh: Churchill Livingstone, 1994: 81941.
  • 4
    Brettler, DB, Levine, PH. Clinical manifestations and therapy of inherited coagulation factor deficiencies. In: Coleman, RW, Hirsh, J, Mader, VJ, Saltzman, EW, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn. Philadephia: J P Lippincott Co., 1994: 16983.
  • 5
    Arun, B, Kessler, CM. Clinical manifestations and therapy of the haemophilias. In: Coleman, RW, Hirsh, J, Mader, VJ, Clowes, AW, George, JN, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Inherited Hemorrhagic Disorders, 4th edn. Philadelphia: J P Lippincott, Williams & Wilkins, 2001: 81524.
  • 6
    The Haemophilia Alliance National Service Specification, Service Standards and Deliveries. Types of haemophilia. http://www.haemophiliaalliance.org.uk 2001.